Self-replicating, self-amplifying probes for detection of disease-related nucleic acid sequences

用于检测疾病相关核酸序列的自我复制、自我扩增探针

• 批准号: 5423648
• 负责人: Professor Dr. Jörg Steffen Hartig
• 金额: --
• 依托单位: Arbeitsgruppe Chemie biologisch funktionaler Materialien
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2004
• 资助国家: 德国
• 起止时间: 2003-12-31 至 2007-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5423648?language=en
• 关键词: Self; replicating; self; amplifying; probes

Methods for the specific detection of nucleic acid sequences are of high importance in modern molecular biology. Enzymatic ligations are often used for diagnostic purposes, although they display some limitations. Depending on the enzymes used, ligation of RNA and modified nucleic acids is often inefficient compared to unmodified DNA. In addition, enzymatic ligation reactions are difficult to carry out in vivo. Although nonenzymatic probes are available, they lack signal amplification. Recently, Kool and co-workers have developed probes consisting of modified DNA, which were used to efficiently detect DNA and RNA target sequences by a self-ligation mechanism with high specificity against single mismatches. This nonenzymatic reaction shows some signal amplification. By utilizing these self-ligation probes, it has been possible to detect Escherichia coli by targeting ribosomal RNA in situ. The proposed research project aims at developing a new class of self-ligating probes for detection of less abundant RNA and even DNA in fixed and living cells. A novel probe design should prevent the common problem of product inhibition, enabling enhanced turnover. Furthermore, by rendering the self-ligation to occur exponentially, increased sensitivity should be gained, enabling detection of traces of pathogenic bacteria as also to sense and localize disease-related DNA and RNA sequences in human cells including single nucleotide polymorphisms, oncogenes and mis-splicing events.

核酸序列的特异性检测方法在现代分子生物学中具有高度重要性。酶连接通常用于诊断目的，尽管它们显示出一些局限性。根据所使用的酶，与未修饰的DNA相比，RNA和修饰的核酸的连接通常是低效的。此外，酶促连接反应难以在体内进行。虽然非酶探针是可用的，但它们缺乏信号放大。最近，Kool及其同事开发了由修饰的DNA组成的探针，其用于通过对单个错配具有高特异性的自连接机制来有效地检测DNA和RNA靶序列。这种非酶促反应显示出一些信号放大。通过利用这些自连接探针，可以通过原位靶向核糖体RNA来检测大肠杆菌。拟议的研究项目旨在开发一类新的自连接探针，用于检测固定和活细胞中丰度较低的RNA甚至DNA。新型探针设计应防止产物抑制的常见问题，从而实现增强的周转。此外，通过使自连接指数地发生，应该获得增加的灵敏度，使得能够检测痕量的病原性细菌，以及还能够感测和定位人类细胞中的疾病相关的DNA和RNA序列，包括单核苷酸多态性、致癌基因和错误剪接事件。